In the health food and biomedical markets there exists a growing demand for extracts of animal products that contain enriched levels of growth factors and other low molecular weight polypeptides. This demand stems from the enhanced bioactivity, and often greater solubility and stability, of the smaller polypeptides relative to the large proteins present in the tissue or other source material. In particular, low molecular weight peptide and growth factor extracts have properties making them suitable for a number of diverse applications including medical (e.g. wound healing) products, ingredients in dietary supplements, cosmetics and cell growth media.
A variety of standard methods for aqueous extraction of animal tissues have been developed and used widely for isolation of proteins, Scopes R.K. (1987). Typically aqueous extraction involves using some method for breaking up cells, such as ultrasound or mechanical disruption (in a blender), in the presence of water or an aqueous salt or buffer. The pH of the extraction system is sometimes manipulated, or detergents and other additives used, in order to enhance the solubility of specific target proteins (e.g. membrane-associated enzymes). However, typically the initial total protein extract will contain proteins having a wide range of molecular weights. Further processing steps are then required for selective enrichment of growth factors and other low molecular weight polypeptides in the total protein extracts.
Commercial production of growth factor-enriched tissue extracts enriched with low molecular weight peptides requires the availability of practical, cost effective methods for removal of unwanted higher molecular weight proteins from the mixtures. Current methods that are applicable on an industrial scale for fractionation of tissue extracts and other animal-derived material (e.g. blood, milk, colostrum), with potential enrichment of growth factors, include:                Ultrafiltration        Gel filtration chromatography (GFC) (also known as size exclusion chromatography)        Other chromatographic fractionation systems (e.g. ion exchange, hydrophobic interaction, affinity)        Liquid phase partitioning in multiphasic systems        Precipitation from aqueous solution (i.e. from the liquid phase) using water miscible organic solvents (ethanol, acetone), typically the organic solvent is ice cold sometimes with the addition of immiscible organic solvents (chloroform) to enhance the denaturing effect, Scopes, R.K. (1987).        Salting out with neutral salts or amino acids        
However, in most instances these methods have been used to isolate enzymes and other moderately large proteins, rather than the concentration of growth factors and other peptides. For example, precipitation using cold ethanol is the basis of the traditional Cohn fractionation method for preparation of albumin and other proteins from plasma.
Consequently, these methods have disadvantages, or are otherwise not well suited, for general enrichment of growth factors and other low molecular weight polypeptides.
Ultrafiltration and GFC both require expensive capital investment, are sensitive to fouling, and the latter results in dilution of the desired low molecular weight fraction. Other chromatographic fractionation systems can also be expensive to operate, and tend to be used to purify specific peptides rather than isolate high or low molecular weight fractions.
Liquid phase partitioning in multiphase systems (e.g. cloud point extraction and aqueous two-phase systems) is particularly of use when a labile protein (e.g. an enzyme) is the desired product as the protein is retained throughout in an aqueous environment, Scopes R.K. (1987), Tani H. et al. (1997). This assists with the retention of the biological activity of the molecule. The disadvantage of the method is that the desired product is obtained in the presence of large amounts of detergents, salts and/or water-soluble polymers which must be removed. This adds to the cost of the process and frequently necessitates the use of additional steps such as ultrafiltration.
The other remaining methods also all result in addition of large amounts of other chemicals (organic solvent, amino acids or salts) which must be removed from the solution of polypeptide. As well as increasing costs due to the need for additional processing steps, these may introduce safety issues as in the case of the use of large volumes of flammable and/or toxic solvents. The safe handling and eventual removal of such solvents requires the use of specially designed, and typically very expensive, processing facilities and equipment.
All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.
It is acknowledged that the term ‘comprise’ may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term ‘comprise’ shall have an inclusive meaning—i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term ‘comprised’ or ‘comprising’ is used in relation to one or more steps in a method or process.
It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.
Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.